PROJECT SUMMARY Salmonellae are Enterobacteriaceae that cause a spectrum of diseases in humans and animals, including enteric (typhoid) fever and gastroenteritis. Typhoid fever, caused primarily by Salmonella enterica serovar Typhi (S. Typhi), results in a life-threatening systemic disease that is responsible for significant morbidity and mortality annually worldwide. Approximately 5% of individuals infected with S. Typhi become chronic carriers with the gallbladder (GB) as the site of persistence. S. Typhi is a human- restricted pathogen, therefore asymptomatic carriers represent a critical reservoir for further spread of disease. We have demonstrated that gallstones (GSs) aid in the development and maintenance of GB carriage in a mouse model (utilizing S. Typhimurium, which causes a typhoid-fever like disease in mice) and in humans, serving as a substrate to which salmonellae attach and form a protective biofilm. Thus, biofilm formation is a key step in the establishment of carriers. Salmonella in biofilms are known to be recalcitrant to antibiotics and host immunity, presenting a challenge for traditional treatment methods. How S. Typhi subverts the initial immune response during biofilm development and establishes chronic infection is poorly understood. Immune escape likely involves extracellular matrix (ECM) components, but the responsible factors are not known. We focus this proposal on curli fibers/pili, surface appendages that are required for S. Typhimurium biofilm formation, mediate attachment and are sensed by pattern recognition receptors. While published reports suggest curli is not produced in S. Typhi, our preliminary data demonstrates that it is produced, but may be regulated differently than in S. Typhimurium. Furthermore, we demonstrate that curli gene expression is enhance ~30-fold in human bile (but not mouse or ox bile). We address our hypotheses by defining genes involved in curli gene expression, specifically those responding to human bile, the factors in human bile mediating gene activation, and the production of curli on GSs from the mouse model and from human carriers in Kenya. Determining the expression of curli during development of persistent infection in this understudied organ, and having the expertise, preliminary results and tools to examine it, will have a strong and sustained influence on the field and will identify key inflection points on which to focus future work.